Molecular regulation of breast cancer metastasis

描述(申请人提供)：我们克隆了一种名为BRMS1的基因，它可以在不抑制致瘤性的情况下抑制六个独立来源的人和小鼠细胞系的转移。我们的目标是确定BRMS1转移抑制的生化机制。 假设1-BRMS1抑制转移需要恢复缝隙连接细胞间通讯(GJIC)。BRMS1基因的转导可恢复GJIC，并改变连接蛋白(Cx)的转录，即Cx43表达上调，Cx32表达下调。目标1将讨论BRMS1表达和GJIC是否都是抑制转移所必需的。BRMS1转基因细胞(转移抑制)将使用siRNA选择性下调BRMS1(Aim 1a)或Cx43(Aim 1b)的表达。目的Lc将检测转移细胞中Cx32的减少是否降低转移潜能，而Aim ld将在BRMS1细胞中重新表达Cx32，并测试转移是否增加。所有转基因细胞都将在体外进行GJIC检测，并在体内进行转移检测(目标1e)。 假设2-BRMS1通过与mSin3：组蛋白脱乙酰酶(HDAC)相互作用抑制肿瘤转移。利用酵母双杂交、共IP和层析技术，我们发现BRMS1与包括mSin3和HDAC在内的大型复合体的组成成分发生了物理上的相互作用。我们将确定哪些BRMSI：HDAC：mSin3复合体(ES)负责抑制转移。AIM 2a将使用FPLC、质谱学、共IP和Y2H来定义BRMS1复合体，并鉴定BRMS1相互作用的蛋白质。AIM 2b将绘制负责特定蛋白质相互作用的BRMS1结构域。目的2c将测试BRMS1通路(1)恢复GJIC的能力，(2)调节HDAC活性的能力，(3)抑制转移的能力。 假设3-Brms1的表达将影响原发乳腺肿瘤的转移。我们建议建立Brms1缺失和Brine1转基因过表达的小鼠，并测试内源性表达是否改变了原发乳腺癌的肿瘤发展和/或转移。目的3a利用条件性Cre-Lox重组技术建立Brmsl缺失型小鼠，并验证Tg：MMTV-PYMT乳腺肿瘤发生转移的几率增加的假说。目的3b将产生乳腺特异和普遍表达的Brms1转基因基因，从而导致Brms1的高表达。Tg：Brmsl小鼠将与Tg：MMTV-PYMT杂交，检验转移潜能将会降低的假设。

DESCRIPTION (provided by applicant): We cloned a gene, BRMS1, which suppresses metastasis in six independently-derived human and murine cell lines without suppressing tumorigenicity. Our objective is to determine the biochemical mechanisms underlying BRMS1 metastasis suppression. Hypothesis 1 - Suppression of metastasis by BRMS1 requires restoration of gap junctional intercellular communication (GJIC). Transfection of BRMS1 restores GJIC along with altered transcription of connexins (Cx), i.e., Cx43 is up-regulated while Cx32 is down-regulated. Aim 1 will address whether BRMS1 expression and GJIC are both required to suppress metastasis. BRMSl-transfected cells (metastasis-suppressed) will have either BRMS1 (Aim la) or Cx43 (Aim lb) expression selectively down-regulated using siRNA. Aim lc will test whether decreased Cx32 in metastatic cells decreases metastatic potential while Aim ld will re-express Cx32 in BRMSl-transfected cells and test whether metastasis increases. All transfectants will be tested for GJIC in vitro and metastasis in vivo (Aim 1e). Hypothesis 2 - BRMSl suppresses metastasis via interactions with mSin3:histone deacetylase (HDAC). Using yeast two-hybrid, co-IP and chromatography, we showed that BRMS1 physically interacts with components of large complexes that include mSin3 and HDAC. We will define which BRMSI:HDAC:mSin3 complex(es) are responsible for metastasis suppression. Aim 2a will use FPLC, mass spectroscopy, co-IP and Y2H to define BRMS1 complexes and identify the BRMS1 interacting proteins. Aim 2b will map BRMS1 domains responsible for specific protein interactions. Aim 2c will test the ability of BRMS1 routto (1) restore GJIC, (2) regulate HDAC activity; and (3) suppress metastasis. Hypothesis 3- Brmsl expression will affect metastasis in autochthonous mammary tumors. We propose to develop Brmsl-null and Brine1 transgenic over-expressing mice and test whether endogenous expression alters tumor development and/or metastasis of autochthonous mammary carcinomas. Aim 3a will generate Brmsl null mice using conditional Cre-Lox recombination and test the hypothesis that the frequency of metastases from tg: MMTV-PyMT mammary tumors will increase when the mice are crossed. Aim 3b will generate mammary-specific and ubiquitous expression Brmsl transgenes that will result in high expression of Brmsl. tg:Brmsl mice will be crossed with tg: MMTV-PYMT and test the hypothesis that metastatic potential will decrease.